Glycine receptors (GlyRs) play a critical role in neuronal excitability in the mammalian brain stem and spinal cord. Their activation reduces the excitability of neurons associated with sensory information, motor control and respiration, functions that are significantly altered during ethanol (EtOH) intoxication. Studies from our and other laboratories demonstrated that the glycine-activated Cl current can be potentiated by low, clinically relevant EtOH concentrations in native neurons and heterologous expression systems. Studies by Aguayo and others have revealed a novel mechanism of EtOH action on GlyRs that involves modulation by G protein activation. We (Aguayo) recently demonstrated that GlyRs are modulated by G protein ?? subunits via basic residue domains within the TM3-4 intracellular loop of a1-containing GlyRs. Importantly, our preliminary results showed that mutants resistant to G??? dimer modulation were insensitive to potentiation by EtOH. Noteworthy, other receptor properties (apparent agonist and antagonist affinities and single channel conductance) were normal and receptor modulation by general anesthetics was not altered, indicating significant selectivity for EtOH. Altogether, these data demonstrate that basic amino acids in the large intracellular loop regulate the sensitivity of GlyRs to physiologically relevant concentrations of EtOH. However, the prime question that has not yet been answered is: What is the importance of G??? modulation of GlyR function in terms of expression of the clinically relevant effects of EtOH exposure on whole animal behavior? To fill this gap in our knowledge, we propose to extend the pioneering studies of Aguayo to the whole animal level. Our working hypothesis is that EtOH affects GlyR function by free G??? through specific regions in the intracellular loop, and that G??? modulation of GlyRs is critical for the behavioral effects of EtOH. Our primary objectives are to generate gene knockin mice that harbor mutant a1 GlyRs that are insensitive to modulation by G???, and test these mice for cellular and behavioral responses to EtOH. [unreadable] [unreadable] [unreadable]